Abstract 1700: Clonal emergence of resistance to methotrexate in osteosarcoma barcoded PDX cells
Authors: Giuseppe Longo, Sylvester Jusu, Zhongting Zhang, Zhaohui Xu, Wendong Zhang, Michael Roth, Rui Yang, Douglas Harrison, Massimo Libra, Amer Najjar, Jonathan Gill, Richard Gorlick
Journal: Cancer Research (2023)

Abstract Introduction: Osteosarcoma is known to have significant intratumoral heterogeneity with branched cancer evolution. We have studied the clonal emergence of resistance to targeted and cytotoxic chemotherapy in osteosarcoma barcoded cell lines derived from patient derived xenograft (PDX) models, in order to verify if short macroevolutionary jumps occur, as a consequence of chromosomal instability, leading to subclonal diversification after chemotherapy treatment. Methods: We have previously shown in our barcoded PDX models that, by applying selective pressure with methotrexate (MTX) IC95 treatment, a clonal subpopulations more resistant to MTX emerges. Preliminary data from Next-Gen sequencing of the genomic DNA extracted from the vehicles and selected clones have shown a wide variability of barcode abundance in the clones and the vehicles. Interestingly a reduced barcode abundance was observed in the clonal subpopulations as compared to the vehicles. Also, a wide range of clonal diversity was observed among the different clones and the vehicles, being high in the M17 PDX and its clones, and low in the OS252 PDX and its derived clones. Functional assays of MTX transport were performed by measuring the cellular uptake of [3H] MTX as previously described (Matherly L. Cancer Res. 1991) in both PDXs and derived clones. Our observations show that [3H] MTX uptake was affected differently in PDXs and derived clones, highlighting a wide variability in MTX transport, possibly due to different mutations in the reduced folate carrier (RFC) gene in different clones. In some of these clones, resistance to MTX might also be due to alterations in other genes involved in MTX cytotoxic activity (DHFR, FPGS, GGH). Results: By applying selective pressure with MTX to PDX derived cell lines we obtained clones whose genomic DNA has been studied with NGS technique. The results obtained by performing the functional assay of MTX transport on both PDXs and derived clones showed a wide variability in the MTX uptake, further supporting the initial hypothesis. Sequencing will allow us to study if there are mutations in the RFC gene and/or in other genes involved in MTX resistance in all the PDXs and derived clones. RNA sequencing (RNA-Seq) will suggest the pathway alterations that may be associated with the emergence of resistance. Conclusion - Barcoded PDX derived osteosarcoma cell lines have been created with resistant clones obtained after MTX treatment. Sequencing of the RFC gene and other genes involved in resistance to MTX, and RNA-Seq profiling will yield critical information on the clonality and mechanisms of resistance which will be presented. Acknowledgments: This work was funded by Swim Across America, the Foster Foundation, and the Barbara Epstein Foundation. Citation Format: Giuseppe Longo, Sylvester Jusu, Zhongting Zhang, Zhaohui Xu, Wendong Zhang, Michael Roth, Rui Yang, Douglas Harrison, Massimo Libra, Amer Najjar, Jonathan Gill, Richard Gorlick. Clonal emergence of resistance to methotrexate in osteosarcoma barcoded PDX cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1700.